Nasopharyngeal carriage and antimicrobial susceptibility profiles of Streptococcus pneumoniae among children with pneumonia and healthy children in Padang, Indonesia

Streptococcus pneumoniae is one of the pathogenic bacteria causing invasive pneumococcal diseases such as pneumonia, sepsis, and meningitis, which are commonly reported in children and adults. In this study, we investigated the nasopharyngeal carriage rates, serotype distribution, and antimicrobial susceptibility profiles of S. pneumoniae among children with pneumonia and healthy children under 5 years old in Padang, West Sumatra, Indonesia. Nasopharyngeal swabs were collected from 65 hospitalized children with pneumonia in a referral hospital and from 65 healthy children at two day-care centers from 2018 to 2019. S. pneumoniae was identified by conventional and molecular methods. Antibiotic susceptibility was performed with the disc diffusion method. Out of 130 children, S. pneumoniae strains were carried by 53% and 9.2 % in healthy children (35/65) and children with pneumonia (6/65), respectively. Serotype 19F was the most common serotype among the isolated strains (21%) followed by 6C (10%), 14, 34 (7 % each), and 1, 23F, 6A, 6B (5 % each). Moreover, 55 % of the strains (23/42) were covered by the 13-valent pneumococcal conjugate vaccine. Most isolates were susceptible to vancomycin (100%), chloramphenicol (93%), clindamycin (76%), erythromycin (71%), and tetracycline (69%). Serotype 19F was commonly found as a multi-drug resistant strain.


INTRODUCTION
Pneumonia, an infection of the lung parenchyma, still remains a major cause of morbidity and mortality in children worldwide [1]. Pneumonia is the leading cause of death for children under 5 years of age that killed 740 180 children (14 % of all deaths of children under 5 years of ages) worldwide in 2019, occurring mainly in developing countries [2]. Pneumonia also results in one of the largest state expenditures both directly, through medical costs and indirectly, by the loss of working hours in the parents taking care of their sick children [3]. In Indonesia, 16 % of children's deaths (aged between 29 days to 11 months) in 2019 were due to pneumonia followed by diarrhoea [4]. Meanwhile, pneumonia (9.5 %) is the second largest cause of death in children between aged 12 to 60 months under five [4]. Streptococcus pneumoniae, as one of the causative agents of pneumonia, is an opportunistic pathogen colonizing the human nasopharynx [5]. This colonization can increase risk of infection depending on the immune system's condition [5,6]. In this study, we investigated the nasopharyngeal carriage rates, serotype distribution, and antimicrobial susceptibility profiles of S. pneumoniae among children with pneumonia and healthy children under 5 years of age in Padang, West Sumatra, Indonesia.

Study design and specimen collection
The study was conducted at the Dr. M. Djamil Hospital, a provincial referral hospital, and at two day-care centres located in Padang, West Sumatra, Indonesia. The children enrolled in the Dr. M. Djamil Hospital were admitted patients with clinical pneumonia from April 2018 to December 2019. The enrollment criteria of children with pneumonia included new or progressive infiltrates on chest radiographs with ≥2 of the following criteria: dyspnoea, cough, hemoptysis, chest pain, and fever occurring ≤14 days before admission. Healthy children under 5 years of age, who were attending day-care centres, were also enrolled from August to December 2019. Demographic characteristics and clinical data were collected and recorded in the case report form.
The Nasopharyngeal (NP) swab specimens were collected from both groups using flocked nylon swabs (Copan; Cat. No. 503CS01) placed into 1 ml skim milk-tryptone-glucose-glycerol (STGG) medium followed by vortex. Samples were then stored at −80 °C. The specimens were regularly shipped to the Eijkman Centre for Molecular Biology (Eijkman Research Centre for Molecular Biology), Jakarta with dry ice for the isolation and identification of S. pneumoniae.

Bacterial identification
The NP swab specimens were enriched by transferring 200 µl of swab-STGG medium into 6 ml enrichment media consisting of 5 ml Todd-Hewitt broth (BD Bacto; Cat. No. 249240) with 0.5 % yeast extract (BD Bacto; Cat. No. 212750) and 1 ml rabbit serum (Gibco; Cat. No. 16120099), and then were incubated at 37 °C with 5 % CO 2 for 5 h. A 10 µl of the enriched specimens was inoculated onto a sheep blood (8%) agar plate (sBAP, comprised of TSA II; BD cat. no. 212 305, with 8 % v/v sheep blood supplementation) and incubated at 37 °C with 5 % CO 2 for 20 h [7]. After that, all blood agar plates were examined for suspected S. pneumoniae colonies with the following colony morphologies: alpha-hemolysis, mucoid, and depressed centre. Suspected colonies were streaked on a sBAP and a disc containing 5 µg of optochin was placed onto the inoculated media. Colonies susceptible to optochin (inhibition zone diameter >14 mm) and positive for bile solubility were identified as S. pneumoniae.
Bacterial DNA was extracted by enzymatic fast DNA extraction as follows: the overnight colony on BAP was resuspended in 300 µl of NaCl 0.85 % and the suspension was vortexed. This mixture was then incubated at 70 °C for 15 min followed by centrifugation at 10 000 r.p.m. for 2 min. The supernatant was discarded and the pellet was then resuspended with 50 µl Tris-EDTA (TE) buffer followed by homogenization. A volume of 8 µl hyaluronidase (30 mg ml −1 ) and 12 µl mutanolysin (2500 U ml −1 ) were added and the suspension was vortexed. The suspension was then incubated for 30 min at 37 °C followed by enzyme inactivation at 100 °C for 10 min. The mixture was then centrifuged at 10 000 r.p.m. for 4 min and the supernatant containing the DNA was stored at −20 °C until further analysis. Serotype determination was performed by a sequential multiplex PCR (SM-PCR) as published by Carvalho et al. followed by Quellung reaction method as described previously [5]. The serogroup six results obtained by PCR was further tested by using enzymatic restriction digest to distinguish serotypes 6A, 6B, 6C, and 6D [8].

RESULTS
The NP swab specimens were collected from a total of 130 children; 65 hospitalized children with pneumonia (mean age of 11.2±10.5 months) during the one and half years period of study, and 65 healthy children (mean age of 29.4±16.1 months) that were recruited from daycares for a period of 4 months. In this study, the proportion of infants less than 1 year of age was 80.0 % (52/65) for the admitted patients and only 13.8 % (9/65) for healthy children. The children's characteristics from both groups are shown in Table 1. The majority of the hospitalized children's clinical symptoms were fever (72.3 %), rhonchi (76.9 %), and cough (66.2 %). Most of the hospitalized children had history of previous antibiotics use (54/65; 83.1 %) and chest X-ray infiltrates (56/65, 86.2 %) ( Table 1). The good nutritional status of healthy children was higher than children with pneumonia (72.3 % vs 53.8 % respectively).
A total of 42 S. pneumoniae strains were isolated from 41 positive NP swab specimens, with a single sample (PDG 98) from one healthy-children simultaneously positive for strains of serotype 14 and 19A (Table S1). Serotype 19F was the most common serotype among the cultured strains (21 %; 9/42) followed by 6C (10 %; 4/42), 14, 34 (three carriers each;  (Fig. 2a). We found that nine isolates (21 %) were untypeable (UNT) strains using the SM-PCR method. Among children with pneumonia, we isolated six S. pneumoniae isolates including serotype 19F and 6B (one strain each) and four UNT strains, while among healthy children, we identified thirty-six S. pneumoniae isolates with serotype 19F as the most prevalent serotype among cultured strains (8/36) (Fig. 2b). In this study, S. pneumoniae strains that were covered by the pneumococcal conjugate vaccine (PCV13) was 50 %.
Furthermore, we found that there were 36 % (13/36) and 67 % (4/6) of S. pneumoniae grouped as multi-drug resistant (MDR) with resistance to ≥3 groups of antibiotics among isolates of S. pneumoniae isolated from healthy children and children with pneumonia respectively (Fig. 4a). The serotype 19F is the most common serotype found as MDR (Fig. 4b, c).

DISCUSSION
In this study, we found that the prevalence of S. pneumoniae in children with pneumonia (9.2 %) was lower than healthy children (53 %) in Padang, West Sumatra, Indonesia. A possible explanation for this was due to previous antibiotic use at the secondary level hospital before being referred to our third level hospital and majority of children with pneumonia symptoms are under 1 year old (80 %). A previous study in Thailand also reported that children with pneumonia showed lower prevalence of S. pneumoniae colonization (54.5 %) compared to community controls (62.5 %) [10]. However, this is in contrast with a previous study in India reporting that the prevalence of S. pneumoniae carriage among children with clinical pneumonia was higher (74.7 %) than community children (54.5 %) [11]. In comparison with a previous study, the range of prevalence of pneumococcal carriage among children under 5 years of age in Indonesia was 13.9-68 % [12]. In addition, a previous study reported that attendance of children in daycare showed significant impact on S. pneumoniae colonization [13]. Another study reported that incidence of pneumonia was higher in malnourished children and is significantly different in children with no malnutrition as previously reported [14]. Furthermore, malnutrition was reported to correlate with increase severity and fatality of pneumonia case incidence [1].
In this study, we discovered that the serotypes circulating in West Sumatra, Padang are dominated by the invasive serotypes currently covered in the pneumococcal vaccine. More than half of isolates are included in the pneumococcal vaccine conjugate. This is also in concordance with previous studies which reported the common serotypes of S. pneumoniae circulating in Indonesia are the vaccine serotypes [15][16][17]. The vaccine serotypes are common serotypes distributed in Indonesia because Indonesia has not included the pneumococcal vaccine as part of the national routine vaccination programme. Among the vaccine serotypes, this study found that serotype 19F is the most frequent serotype identified. The frequency of each serotype colonizing children's nasopharynx is found to vary across regions in Indonesia. In Kotabaru, Kalimantan and Lombok, West Nusa Tenggara, the most common serotype is 6A/6B [15,17]. Meanwhile in Bandung, West Java, the 15B/15C is the most common serotype found in healthy children's nasopharynx.
This study also found that most of the isolates are resistant to co-trimoxazole and oxacillin which is also in concordance with recent publications reporting most of the S. pneumoniae isolates as being resistant to co-trimoxazole [17-19]. Moreover, we   discovered that not only is serotype 19F the most common serotype but is also resistant to more than three groups of antibiotics, identifying serotype 19F as MDR. This serotype has also been reported as the most common serotype with MDR characteristics in recent studies [17,20].
The limitations of this study are related to the status of the hospital as the tertiary hospital and a referral centre, in which the history of antibiotic administration could not be provided for this study. In addition, in this study, the hospitalized children with pneumonia group was mostly children under 1 year of age, while in the healthy group there were more children over 1 year of age. This might affect the results of the carriage rates of S. pneumoniae. Children under 1 year of age are not common in day-care centres compared to children over 1 year of age, causing the healthy group to have varying results of colonization due to the high risk of pneumococcal transmission. In conclusion, prevalence of pneumococcal carriage in hospitalized children with pneumonia is lower than healthy children under 5 years old in Padang, Indonesia and serotype 19F was commonly found as a multi-drug resistant strain. Find out more and submit your article at microbiologyresearch.org.

Peer review history
Reviewer 1 Comments to Author: This manuscript presents data analysing the carriage of S.pneumoniaein a cohort of healthy children vs those with pneumonia, from a tertiary care hospital in Indonesia. The study focuses on characterising the strains isolated from this cohort and their resistance to anti-microbials. There are no apparent ethical or conflict-of-interest concerns, and the methods for the isolation, Response:TE buffer is Tris-EDTA Buffer. We have added the information in the line 121. 120 -Make the rpm number the same as previous style.

Response: we have revised the rpm number (Line 125)
130-133 -These should be concentrations rather than weights.
Response: As stated in CLSI Guidelines, for disk diffusion the antibiotic amount used is in wight of antibiotics (in microgram) as well as in the insert package of antibiotic disk we used.
130 -Reference for the method.  Table 1-Numbers don't always add up to 65 or the full cohort number, but no explanation provided.
Response: Thank you for your comments. We confirmed that number of participants = 130 children; children with pneumonia = 65 and healthy children = 65. We revised the text.
We found that one NP swab specimen (PDG 98) from one healthy-children positive simultaneously for strains of serotype 14 and 19A (Line 169-171) Figure 1 -Y-axis should extend to 100% and axis line is missing. Y-axis label should be clearer e.g. % of children. No stats provided on the graphs, and blue bars e.g. hospitalised patients 13-24 months looks about double that of the other two age brackets -the results text mentions that there is a difference but no statistical analysis to back it up.
Response: Thank you for your suggestion. We modified the Figure 1 and the text as below "The carriage rates were 7.7%, 20.0%, and 12.5% for the aged group of less than one year of age (4/52), the aged group of 13 to 24 months (1/5), and the aged group of 25 to 60 months (1/8), respectively. Meanwhile, among healthy children, the S. pneumoniae carriage were 55.6%, 54.2%, and 53.1% for for the aged group of less than one year of age (5/9), the aged group of 13 to 24 months (13/24), and the aged group of 25 to 60 months (17/32), respectively ( Figure 1). " Line 161-167.     180 -182-confusing wording, state that the strains are less susceptible to these compounds rather than the compounds have been less susceptible.
Response: Yes, you are correct. The statement should describe the resistance profile of S. pneumoniaeisolates against the compounds/antimicrobials.

Response: we have added the information in line 194
Figure4a -Might be helpful to state which antibiotics in figure legend, Y-axis has no line, Y-axis needs label. X-axis label could be clearer e.g. number of antibiotics resistant per strain.

Response:
We have revised the graph as your suggestions. We have added the Y-axis label and revised the X-axis label.

-Change commonest to most common
Response:we have replaced the commonest to most common.

223-224 -Clarify that this is a test to diagnose pneumonia and the 90.3% was correct identification
Response: Correct, the chest X-ray is used to diagnose pneumonia.
234 -Change to "is found to vary" rather than is found vary Response: We have revised as your suggestion.

Response:
We have changed the commonest to the most common.
252-Ampicillin and gentamycin is mentioned but previous history of antibiotic administration could not be provided. clarify that this is inferred based on common treatment routine.
Response: Thank you for your suggestion. We removed it.

309-310 -Reference 4 style is not consistent with other references
Response: We revised it.
248-250 -Children under 1 year of age less likely to be in day-care? Which may explain some results e.g. older children more likely to be exposed to S. pneumoniae.
Response: This might explain the different rate of colonization between 2 groups. We have added your suggestion into manuscript line 249-252.

Reviewer #2
Reviewer 2 Comments to Author: In this manuscript, the authors set out to address a lack of information on the carriage of Streptococcus pneumoniae, a causative agent of pneumonia, in healthy and hospitalized children in Indonesia. They also attempt to identify the serotypes circulating in these cohorts and test their susceptibility to antimicrobials.
I have no major concerns about the manuscript -the methods are well articulated and the authors have done what they set out what they attempted to do, namely: describe the prevalence, serotype frequency, and antimicrobial susceptibilities of S.pneumoniae in cohorts of healthy and hospitalised children in the Padang region of Indonesia. They ultimately find that few hospitalised children actually carry S.pneumoniae in the nasopharynx, identify the most common serotype of S.pneumoniae found in the these cohorts, and provide support for a recent study suggesting that S.pneumoniae is commonly resistant to a commonly used antimicrobial (co-trimoxazole). There are significant age differences between the hospitalized and healthy cohorts that could account for differences in S.pneumoniae prevalence but the authors acknowledge and discuss this in the manuscript. They also discuss their findings in relation to other studies reporting S.pneumoniae carriage and antimicrobial susceptibility in the wider region.
I only have a few minor comments, most of which concern the formatting and presentation of the manuscripts figures. While the manuscript is generally well written, there are a few sentences key to understanding the results of the study that could be made clearer grammatically -I've highlighted a few below but the journal editor may be able to provide further assistance with respect to this.

Minor Comments -Figures
Generally, the formatting across figures could be more consistent i.e. font size, axis size etc.

Response:
We have revised all figures and made all figures similar.       Response: thank you for your suggestion, we will make the information similar as number of isolates (n)

Minor Comments -Manuscript
Line 27 -in children and adults.
Response: thank you for your suggestion, we have added the information.
Line 27-28 -the authors say "Streptococcus pneumoniae commonly causes pneumonia in children" and with the next sentence say there is a lack of reports for Streptococcus pneumoniae causing pneumonia in children. Do they mean there is a lack of information on whether particular serotypes are responsible?
Response: Correct, we have revised the sentence to include the serotypes.

Response: we have revised the commas to decimals
Line 41 -most isolates.
Response: we have revised the manuscript accordingly.
Line 62 -as one of the.

Response:
we have revised the manuscript as suggested.
Line 95 -the company producing nylon swabs should be included.
Response: we have added the company name.
Line 176 -the majority are not susceptible to oxacillin and co-trimoxazole.
Response: we have revised the manuscript according to your suggestion.
Line 177-179 -I think this would be clearer if stated that all isolates were resistant to oxacillin and co-trimoxazole rather than © 2023 Anonymous. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.

Date report received: 22 March 2023 Recommendation: Minor Amendment
Comments: In this manuscript, the authors set out to address a lack of information on the carriage of Streptococcus pneumoniae, a causative agent of pneumonia, in healthy and hospitalized children in Indonesia. They also attempt to identify the serotypes circulating in these cohorts and test their susceptibility to antimicrobials. I have no major concerns about the manuscript -the methods are well articulated and the authors have done what they set out what they attempted to do, namely: describe the prevalence, serotype frequency, and antimicrobial susceptibilities of S.pneumoniae in cohorts of healthy and hospitalised children in the Padang region of Indonesia. They ultimately find that few hospitalised children actually carry S.pneumoniae in the nasopharynx, identify the most common serotype of S.pneumoniae found in the these cohorts, and provide support for a recent study suggesting that S.pneumoniae is commonly resistant to a commonly used antimicrobial (co-trimoxazole). There are significant age differences between the hospitalized and healthy cohorts that could account for differences in S.pneumoniae prevalence but the authors acknowledge and discuss this in the manuscript. They also discuss their findings in relation to other studies reporting S.pneumoniae carriage and antimicrobial susceptibility in the wider region. I only have a few minor comments, most of which concern the formatting and presentation of the manuscripts figures. While the manuscript is generally well written, there are a few sentences key to understanding the results of the study that could be made clearer grammatically -I've highlighted a few below but the journal editor may be able to provide further assistance with respect to this. Minor Comments - Figures Figure 4 -Y-axis could have either number of isolates or isolates (n). Minor Comments -Manuscript Line 27 -in children and adults. Line 27-28 -the authors say "Streptococcus pneumoniae commonly causes pneumonia in children" and with the next sentence say there is a lack of reports for Streptococcus pneumoniae causing pneumonia in children. Do they mean there is a lack of information on whether particular serotypes are responsible? Line 37should be 9.2% not 9,2%. Line 41 -most isolates. Line 62 -as one of the. Line 95 -the company producing nylon swabs should be included. Line 176 -the majority are not susceptible to oxacillin and co-trimoxazole. Line 177-179 -I think this would be clearer if stated that all isolates were resistant to oxacillin and co-trimoxazole rather than 'less susceptible'. Line 177-179 -Not sure what "tetracycline (100%)" means here…around half of the isolates in this data appear to be susceptible to tetracycline? 179 -183 -I think this needs to be more carefully worded to make it clear that "less susceptible" means fewer strains that are susceptible to the antibiotic. "vancomycin is found susceptible for all isolates is also slightly confusing -something like "All isolates from healthy children and children with pneumonia are susceptible to vancomycin" might make this clearer. Line 188 -most common. Line 204 -symptoms. Line 205 -Another explanation could be an age effect? Most children with pneumonia were under the age of 1 and most healthy children were older? Line 207 -S.pneumoniae should be italicised. Line 215 -malnourished. Line 214 -216 -I would avoid using "this study" as readers might assume the results discussed in this sentence were produced by the authors and not in reference to another study.  Table 1-Numbers don't always add up to 65 or the full cohort number, but no explanation provided. Figure 1 -Y-axis should extend to 100% and axis line is missing. Y-axis label should be clearer e.g. % of children. No stats provided on the graphs, and blue bars e.g. hospitalised patients 13-24 months looks about double that of the other two age brackets -the results text mentions that there is a difference but no statistical analysis to back it up. Figure 1 legend -S. pneumoniae Figure 2a -Y-axis should extend to 100%, no line for Y-axis, Y-axis label should be clearer. shows. 180 -182-confusing wording, state that the strains are less susceptible to these compounds rather than the compounds have been less susceptible. 186 -Explain what MDR is an abbreviation of. Figure4a -Might be helpful to state which antibiotics in figure legend, Y-axis has no line, Y-axis needs label. X-axis label could be clearer e.g. number of antibiotics resistant per strain. 188 -Change commonest to most common Figure 4b, 4c -Key should be clearer e.g. state that this is number of antibiotics in the graph, Y-axis has no line. 208 -Change comma in 54,5% to decimal point, same with 62,5% 212 -Change comma in 13,9 to decimal point 223-224 -Clarify that this is a test to diagnose pneumonia and the 90.3% was correct identification 234 -Change to "is found to vary" rather than is found vary 235 + 236 + 244-Change commonest to most common. 252-Ampicillin and gentamycin is mentioned but previous history of antibiotic administration could not be provided. clarify that this is inferred based on common treatment routine. 309-310 -Reference 4 style is not consistent with other references 248-250 -Children under 1 year of age less likely to be in day-care? Which may explain some results e.g. older children more likely to be exposed to S.pneumoniae.

Please rate the manuscript for methodological rigour Satisfactory
Please rate the quality of the presentation and structure of the manuscript Satisfactory